Our understanding of substance addiction may be considerably increased if we know how neurotransmitter levels vary in select regions of cerebral cortex. In particular, it is believed that using addictive drug alters attentional processing mediated by acetylcholine (ACh). However, further progress in ACh research in the normal and addicted brain is hampered by the shortcomings of current techniques to measure levels of ACh, as these methods have limited time resolution and spatial accuracy. To overcome these problems, we are developing a novel cell-based optical method to detect ACh in vivo. "Sentinel Cells" are engineered cells that express cloned ACh receptors together with a genetically encoded fluorescent probe that reports the activity of the receptors. Sentinel Cells are implanted in rat cerebral cortex and their optical signal detected by two-photon laser scanning microscopy, allowing these cells to be imaged several hundred of micrometers inside the brain. A preliminary realization with HEK293 cells as a substrate validates this approach. We utilize the Gq family of G-protein metabotropic receptors, which couples to the IP3 pathway, and express the M1 G-coupled receptor for ACh together with cytoplasmic expression of a Troponin-C-based calcium sensor. These cells are engineered to respond to heightened extracellular Ach levels via IP3-mediated release of Ca2+ from the endoplasmic reticulum into the cytoplasm, which leads to a change in the extent of fluorescent resonant energy transfer between cyan and yellow fluorescent proteins. In vitro data suggest an effective Kd of ~ 30 nM and a response time of ~ 1 second for our preliminary realization. Complementary in vivo data shows that cells injected into cortex can respond to Ach release from intrinsic cholinergic fibers. Our plan is, first, to continue the development and optimization of Sentinel Cells for the detection of Ach in acute in vivo preparations. Second, to extend the realization of Sentinel cells to fibriblasts and chronic in vivo measurements. Third, to extend the concept of Sentinel Cells with metabotopic receptors to other cell signaling molecules common to cortex. This includes the neurotransmitter gamma-amino butyric acid (GABA) and the neurovascular signaling molecules Neuropeptide Y (NPY), vasointestinal peptide (VIP), and somatostatin (SOM). In toto, Sentinel Cells provide a novel means for (near) real-time monitoring in drug abuse research and basic research in cortical physiology. We propose to develop a novel optical bioassay to detect extracellular signaling molecules in the live brain. This technique combines engineered cells expressing a transmembrane receptor and a fluorescent reporter with in vivo two-photon laser scanning microscopy. The assay has wide ranging applications, including, but not limited to research on abused substances. [unreadable] [unreadable] [unreadable]